Wash buffer kit

RNA labeling and array hybridization was according to Exiqon’s manual. After quality control, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling by following steps: 1μLRNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C. The Reaction was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°CTerminated by incubation for 15 min at 65°C. After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual. The total 25 μL mixture from Hy3™-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min. Then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System-Nimblegen Systems, Inc., Madison, WI, USA). Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon). Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).

The microarray analysis for miRNA profiling was conducted by the Shanghai Kangcheng Technology using the miRCURY LNA Array system (Exiqon, Vedbaek, Denmark). Total RNA was extracted and purified using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. The miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer's guideline for miRNA labeling. After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURYTM LNA Array (v.19.0) (Exiqon) according to array manual. Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon). Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs between two groups were identified through Fold change (>2) and P-value (<0.05).

The total RNA extracted from cells was further purified using an RNeasy Mini Spin Column Kit (Qiagen, Inc., Valencia, CA, USA). After being checked for quality, the RNA samples were labeled using reagents in a miRCURY™ Power Labeling Kit (Exiqon, Denmark) according to the manufacturer's instructions. The labeled RNA samples were hybridized onto a miRCURYTM LNA Array (Exiqon, Denmark), washed with a Wash Buffer Kit (Exiqon, Denmark) and then scanned with a GenePix 4000B microarray scanner (Axon Molecular Devices, San Jose, CA, USA).